SUMMARY Streptococcus pneumoniae is the most common bacterial respiratory pathogen in human immunodeficiency virus (HIV)-infected individuals. The incidence of invasive pneumococcal disease (IPD) is significantly increased, 35-100 fold, in the HIV-positive population. Although vaccination with the pneumococcal polysaccharide vaccine (PPV) is recommended for all those >2 years of age infected with HIV, the response to the vaccine is less than optimal and correlates with the degree of immune suppression as measured by CD4+ T-lymphocyte count (1-10). In addition, vaccine recommendations in newly-diagnosed HIV-positive persons with CD4 counts<200 and those concerning revaccination after 5 years are controversial as there is no evidence to confirm clinical or serological benefit. The poor immune response to vaccine antigens is likely related to the severe B cell dysfunction noted early in HIV disease. The viral protein, gp120, acts as a super- antigen restricted to recognition of the variable heavy chain 3 (VH3) gene segments (11). Moreover, gp120 activates proliferation and differentiation of B cells expressing the VH3 gene (11-14) resulting in progressive deletion of VH3-expressing B cells (15). Depletion of the VH3 expressing B cell population is highly significant as the VH3 gene family products encode >90% of anti-pneumococcal polysaccharide (PPS) antibodies (16- 20). Several studies, using monoclonal anti-idiotypic reagents (21) and PCR-ELISA (22) have demonstrated that HIV-infected individuals generate significantly lower PPS-specific VH3-positive antibody responses compared to HIV-negative controls. A comprehensive study, directly linking anti-PPS antibody levels, functionality and molecular structure/gene family usage, has not been performed. Moreover, the degree of B cell immune reconstitution achieved with highly active anti-retroviral therapy (HAART) is controversial and poorly defined. We have modified a novel technique of single antigen-specific B cell isolation/culture allowing analysis of paired variable heavy (VH) and variable light (VL) gene usage and successfully applied this technique to PPS-specific B cells. In addition, we have recently developed a flow analysis technique that allows us to define the B cell subsets of PPS-responding B cells. This unique ability allows us to define the presence of memory B cells amongst PPS-responding B cell populations in HIV-negative, HIV-positive HAART-na[unreadable]ve and HIV-positive HAART-treated populations. We therefore propose to perform a comprehensive study of the immune response to PPV in HIV-positive individuals. In Specific Aim 1, we propose to define the immune response to PPV in various stages of untreated HIV infection on a quantitative, functional and molecular level, using a novel, single antigen-specific B cell isolation and culture technique. In Specific Aim 2, we will test the hypothesis that individuals on long term HAART therapy are capable of responding to the pneumococcal vaccine and investigate the nature of this response. In Specific Aim 3, we will study the percentage of PPS-specific B cells that are IgM or switched memory B cells in the HIV-negative population and compare this to HIV-positive, HAART-na[unreadable]ve and HIV-positive HAART-treated population following primary vaccination with PPV.